Methods of inhibiting cancer stem cells with hmga1 inhibitors

ABSTRACT

The presently disclosed subject matter relates to methods of inhibiting cancer stem cells and growth of aggressive and/or poorly differentiated metastatic tumors comprising the cancer stem cells with HMGA1 inhibitors. The presently disclosed subject matter also provides methods of selecting and treating a subject with aggressive and/or poorly differentiated metastatic cancer using HMGA1 inhibitors.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/987,264, filed May 1, 2014, which is incorporated herein by referencein its entirety.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under CA149550 awardedby the National Institutes of Health (NIH). The government has certainrights in the invention.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

This application contains a sequence listing. It has been submittedelectronically via EFS-Web as an ASCII text file entitled“111232-00396_ST25.txt”. The sequence listing is 390 bytes in size, andwas created on Apr. 29, 2015. It is hereby incorporated by reference inits entirety.

FIELD OF INVENTION

The presently disclosed subject matter relates to the field of molecularbiology, and particularly to methods of inhibiting cancer stem cells andgrowth of aggressive and/or poorly differentiated metastatic tumors withHGMA1 inhibitors.

BACKGROUND

Despite advances in the ability to detect and treat breast cancer, itremains a leading cause of death in women with cancer, and the incidenceis rising (Siegel et al. (2013) CA Cancer J. Clin. 63:11-30).Approximately 15-20% of all cases are classified as triple-negativebreast cancer, a subtype that is frequently associated with rapidprogression and poor outcomes (Siegel et al. (2013) CA Cancer J Clin.63:11-30; Lee et al. (2010) Cancer Biol. Ther. 9:1017-1024).Triple-negative breast cancer refers to the lack of detectable markersfor the estrogen receptor (ER), progesterone receptor (PR), and Her2/neuamplification.

Treatment of patients with triple-negative breast cancer has beenchallenging due to the heterogeneity of the disease and the absence ofwell-defined molecular targets (Pegram et al. (1998) J. Clin. Oncol.16:2659-2671; Wiggans et al. (1979) Cancer Chemother. Pharmacol.3:45-48; Carey et al. (2007) Clin. Cancer Res. 13:2329-2334).Triple-negative breast cancer tumors are generally larger in size, areof higher grade, have lymph node involvement at diagnosis, and arebiologically more aggressive than other types of breast cancer tumors(Haffty (2006) J. Clin. Oncol. 24:5652-5657). Despite having higherrates of clinical response to presurgical (neoadjuvant) chemotherapy,triple-negative breast cancer patients have a higher rate of distantrecurrence and a poorer prognosis than women with other breast cancersubtypes (Haffty (2006) J. Clin. Oncol. 24:5652-5657; Dent et al. (2007)Clin. Cancer Res. 13:4429-4434). These tumors do not respond to the mosteffective and least toxic therapies, including hormonal therapy(tamoxifen) or herceptin. Less than 30% of women with metastatictriple-negative breast cancer survive 5 years, and almost all die oftheir disease despite adjuvant chemotherapy, which is the mainstay oftreatment (Dent et al. (2007) Clin. Cancer Res. 13:4429-4434).

SUMMARY

The presently disclosed subject matter relates to methods of inhibitingcancer stem cells using HMGA1 inhibitors. In an aspect, the presentlydisclosed subject matter provides a method of inhibiting at least onecancer stem cell, the method comprising contacting the at least onecancer stem cell with an effective amount of at least one HMGA1inhibitor. In some embodiments, inhibiting the at least one cancer stemcell is selected from the group consisting of: i) inhibitingproliferation of the at least one cancer stem cell; ii) inhibitingself-renewal of the at least one cancer stem cell; iii) inhibitinganchorage-independent growth of the at least one cancer stem cell; iv)inhibiting migration of the at least one cancer stem cell; v) inhibitinginvasion of the at least one cancer stem cell; vi) reprogramming the atleast one cancer stem cell from a stem-like state that is refractory toapoptosis to a non stem-like state that is susceptible to apoptosis; andvii) combinations thereof In some embodiments, at least one cancer stemcell is in an aggressive and/or poorly differentiated metastatic tumor,and inhibiting the at least one cancer stem cell inhibits at least oneof: i) growth of the aggressive and/or poorly differentiated metastatictumor; ii) proliferation of the aggressive and/or poorly differentiatedmetastatic tumor; iii) migration of the aggressive and/or poorlydifferentiated metastatic tumor; iv) invasion of the aggressive and/orpoorly differentiated metastatic tumor; v) initiation of new aggressiveand/or poorly differentiated metastatic tumors; and vi) combinationsthereof. In some embodiments, at least one cancer stem cell expressesgreater levels of HMGA1 as compared to non-stem cancer cells in theaggressive and/or poorly differentiated metastatic tumor. In someembodiments, at least one cancer stem cell is selected from the groupconsisting of a triple-negative breast cancer cell, a pancreatic ductaladenocarcinoma cell, a colorectal cancer cell, and a leukemia cell. Insome embodiments, at least one HMGA1 inhibitor reduces the expressionlevel and/or activity of HMGA1. In some embodiments, at least one HMG1inhibitor is an RNA interfering agent. In some embodiments, at least oneHMGA1 inhibitor is an shRNA. In some embodiments, the shRNA targets thenucleotide sequence of SEQ ID NO:1. In some embodiments, the methodincludes contacting the at least one cancer stem cell with achemotherapeutic agent. In some embodiments, the chemotherapeutic agentis gemcitabine. In some embodiments, at least one cancer stem cell iscontacted in a subject. In some embodiments, the subject is a humansubject.

In certain aspects, the presently disclosed subject matter provides amethod of treating an aggressive and/or poorly differentiated metastaticcancer in a subject in need thereof comprising administering atherapeutically effective amount of at least one HMGA1 inhibitor to thesubject. In some embodiments, the aggressive and/or poorlydifferentiated metastatic cancer is selected from the group consistingof triple-negative breast cancer, pancreatic ductal adenocarcinoma cell,colorectal cancer cell, and leukemia. In some embodiments, theaggressive and/or poorly differentiated metastatic cancer comprises atleast one cancer stem cell that overexpresses HMGA1 protein. In someembodiments, the method includes selecting the subject for treatment ofthe aggressive and/or poorly differentiated metastatic cancer with theat least one HMGA1 inhibitor. In some embodiments, selecting the subjectfor treatment of the aggressive and/or poorly differentiated metastaticcancer comprises: (i) obtaining a biological sample comprising cellsfrom the aggressive and/or poorly differentiated metastatic cancer; (ii)assaying the level of HMGA1 expression in the cells from the aggressiveand/or poorly differentiated metastatic cancer; (iii) comparing thelevel of HMGA1 expression in the cells to the level of HMGA1 expressionin a normal control cell; and (iv) selecting the subject for treatmentof the aggressive and/or poorly differentiated metastatic cancer withthe at least one HMGA1 inhibitor if the level of HMGA1 expression in thecells is greater than the level of HMGA1 expression in the normalcontrol cell. In some embodiments, the biological sample is selectedfrom the group consisting of a breast tissue sample, a pancreatic tissuesample, a colon tissue sample, and a bone marrow tissue sample. In someembodiments, at least some of the cells from the aggressive and/orpoorly differentiated metastatic cancer comprise cancer stem cells. Insome embodiments, the at least one HMGA1 inhibitor reduces theexpression level and/or activity of HMGA1. In some embodiments, the atleast one HMGA1 inhibitor is formulated for delivery in a nanoparticle.In some embodiments, the at least one HMG1 inhibitor is an RNAinterfering agent. In some embodiments, the at least one HMGA1 inhibitoris an shRNA. In some embodiments, the shRNA targets the nucleotidesequence of SEQ ID NO:1. In some embodiments, the shRNA is formulatedfor delivery in a nanoparticle. In some embodiments, the subject is ahuman subject. In some embodiments, the method includes administering aneffective amount of a chemotherapeutic agent to the subject. In someembodiments, the chemotherapeutic agent is gemcitabine.

In other aspects, the presently disclosed subject matter provides amethod of selecting a subject with an aggressive and/or poorlydifferentiated metastatic cancer for treatment with at least one HMGA1inhibitor, the method comprising: (a) obtaining a biological sample fromthe subject, wherein the biological sample comprises cells from theaggressive and/or poorly differentiated metastatic cancer; (b)determining the level of expression of HMGA1 in the biological sample;(c) comparing the level of expression of HMGA1 in the biological samplewith the level of expression of HMGA1 in a control sample; and (d)selecting the subject with aggressive and/or poorly differentiatedmetastatic cancer for treatment with at least one HMGA1 inhibitor whenthe level of HMGA1 expression in the biological sample is greater thanthe level of HMGA1 expression in the control sample. In someembodiments, the biological sample is selected from the group consistingof a breast tissue sample, a pancreatic tissue sample, a colon tissuesample, and a bone marrow tissue sample. In some embodiments, theaggressive and/or poorly differentiated metastatic cancer is selectedfrom the group consisting of triple-negative breast cancer, pancreaticductal adenocarcinoma cell, colorectal cancer cell, and leukemia. Insome embodiments, subject is a human subject. In some embodiments, themethod includes treating the subject with aggressive and/or poorlydifferentiated metastatic cancer by administering an effective amount ofat least one HMGA1 inhibitor (e.g., an shRNA, e.g., targeting SEQ IDNO:1) to the subject. In some embodiments, the method includesadministering an effective amount of at least one chemotherapeutic agentto the subject.

Certain aspects of the presently disclosed subject matter having beenstated hereinabove, which are addressed in whole or in part by thepresently disclosed subject matter, other aspects will become evident asthe description proceeds when taken in connection with the accompanyingExamples and Figures as best described herein below.

The practice of the present invention will typically employ, unlessotherwise indicated, conventional techniques of cell biology, cellculture, molecular biology, transgenic biology, microbiology,recombinant nucleic acid (e.g., DNA) technology, immunology, and RNAinterference (RNAi) which are within the skill of the art. Non-limitingdescriptions of certain of these techniques are found in the followingpublications: Ausubel, F., et al., (eds.), Current Protocols inMolecular Biology, Current Protocols in Immunology, Current Protocols inProtein Science, and Current Protocols in Cell Biology, all John Wiley &Sons, N.Y., edition as of December 2008; Sambrook, Russell, andSambrook, Molecular Cloning: A Laboratory Manual, 3rd ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, 2001; Harlow, E. and Lane,D., Antibodies—A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, 1988; Freshney, R. I., “Culture of Animal Cells, AManual of Basic Technique”, 5th ed., John Wiley & Sons, Hoboken, N.J.,2005. Non-limiting information regarding therapeutic agents and humandiseases is found in Goodman and Gilman's The Pharmacological Basis ofTherapeutics, 11th Ed., McGraw Hill, 2005, Katzung, B. (ed.) Basic andClinical Pharmacology, McGraw-Hill/Appleton & Lange; 10th ed. (2006) or11th edition (July 2009). Non-limiting information regarding genes andgenetic disorders is found in McKusick, V. A.: Mendelian Inheritance inMan. A Catalog of Human Genes and Genetic Disorders. Baltimore: JohnsHopkins University Press, 1998 (12th edition) or the more recent onlinedatabase: Online Mendelian Inheritance in Man, OMIMTM. McKusick-NathansInstitute of Genetic Medicine, Johns Hopkins University (Baltimore, Md.)and National Center for Biotechnology Information, National Library ofMedicine (Bethesda, Md.), as of May 1, 2010, World Wide Web URL:http://www.ncbi.nlm.nih.gov/omim/ and in Online Mendelian Inheritance inAnimals (OMIA), a database of genes, inherited disorders and traits inanimal species (other than human and mouse), athttp://omia.angis.org.au/contact.shtml. All patents, patentapplications, and other publications (e.g., scientific articles, books,websites, and databases) mentioned herein are incorporated by referencein their entirety. In case of a conflict between the specification andany of the incorporated references, the specification (including anyamendments thereof, which may be based on an incorporated reference),shall control. Standard art-accepted meanings of terms are used hereinunless indicated otherwise. Standard abbreviations for various terms areused herein.

BRIEF DESCRIPTION OF THE FIGURES

Having thus described the presently disclosed subject matter in generalterms, reference will now be made to the accompanying Figures, which arenot necessarily drawn to scale, and wherein:

FIG. 1A, FIG. 1B, FIG. 1C, FIG. 1D and FIG. 1E show that silencing HMGA1expression halts cell growth and induces dramatic changes in cellmorphology and gene expression: FIG. 1A) lentiviral-mediated delivery ofshRNA to HMGA1 (denoted shHMGA1) results in a marked decrease in HMGA1mRNA and protein in triple-negative breast cancer cell lines(MDA-MB-231, Hs578T); FIG. 1B) proliferation is disrupted in cancer celllines following silencing of HMGA1; FIG. 1C) mesenchymal,fibroblast-like cancer cells undergo dramatic morphologic changes within4 days after treatment with shHMGA1; striking changes were observed inMDA-MB-231 (top panels) and Hs578T cells (bottom panels); bar: 50 um;FIG. 1D) alterations in EMT genes with silencing of HMGA1; and FIG. 1E)migration and invasion is decreased with silencing of HMGA1. *P<0.05;**P<0.01;

FIG. 2A, FIG. 2B, FIG. 2C and FIG. 2D show that silencing HMGA1interferes with orthotopic tumorigenicity and metastatic progression:FIG. 2A) silencing HMGA1 impairs orthotopic tumorigenicity; tumorvolumes+standard deviations are shown; no tumors formed from shHMGA1cells when 10⁴ cells were implanted (for injections with 10⁴ cells, n=3for control or shHMGA1 cells; for injections with 10⁵ cells, n=5 forcontrol and n=8 for shHMGA1 cells; and for injections with 10⁷ cells,n=3 for control and shHMGA1 cells; FIG. 2B) metastatic progression isalmost completely abrogated in cells that do not express HMGA1; thisgraph shows the number metastatic foci to the lung 5 weeks followingimplantation of MDA-MB-23 1 cells (10⁷) into mammary fat pads followingtreatment with control shRNA or shHMGA1; FIG. 2C) the top photographsshow the lungs 8 weeks following implantation into mammary fat pads;there are coalescing sheets of metastatic tumor cells in the lungs ofmice injected with control cells (left) as compared to mice injectedwith shHMGA1 cells (right); due to the widespread tumor cells,individual foci could not be counted; bar: 50 um; and FIG. 2D) thebottom panels show multiple, discreet foci in the lungs 5 weeksfollowing implantation of control cells into mammary fat pads (left) ascompared to mice injected with shHMGA1 cells (right). *P<0.05;**P<0.0001;

FIG. 3A, FIG. 3B and FIG. 3C show that silencing HMGA1 blocksmammosphere formation and depletes tumor-initiator cells: FIG. 3A)silencing HMGA1 blocks mammosphere formation in MDA-MB-231 cells (1°,2°, 3°) and Hs578T cells (1°); FIG. 3B) photographs of mammospheresfollowing treatment of breast cancer cells with control or shHMGA1;silencing HMGA1 significantly inhibits mammosphere formation inMDA-MB-231 and Hs578T cells; bars: 200 um (large panels) and 50 um(insets); and FIG. 3C) tumor numbers at limiting dilutions show thatsilencing HMGA1 depletes the tumor initiator/cancer stem cells inMDA-MB-231 cells; note that no tumors formed following injection of 10⁴cells treated with shHMGA1, while tumors formed in all cases whencontrol cells were injected; both tumor frequency and tumor volumes(±standard deviations) are shown. *P<0.05; **P<0.01;

FIG. 4A and FIG. 4B show that the HMGA1 signature is enriched inpluripotent stem cells, including embryonic and induced pluripotent stemcells: FIG.

4A shows the HMGA signature derived from genes with the greatestexpression changes in the control versus HMGA1 knock-down cellsdisplayed as a heat map. Green depicts down-regulation in expression,while red depicts up-regulation; black denotes little or no change inexpression. The HMGA1 signature overlaps with pluripotent stem cellgenes that distinguish human embryonic stem cells (hESCs) and inducedpluripotent stem cells (iPSCs) from fibroblasts and embryoid bodies(EB). Genes (n=63) were selected for the greatest changes in expressionin the breast cancer cell lines with HMGA1 knock-down as compared to thecontrol breast cancer lines (FIG. 5). In a hierarchical clustering offibroblasts, hESCs, iPSCs, and EBs derived from the hESCs, these genesdistinguish samples by type. The majority of the HMGA1 signature genes,represented in blue along the left margin, are significantlydifferentially expressed between fibroblasts and human pluripotent stemcells (hESC/iPSCs; p<0.001); and FIG. 4B shows a HMGA1 network derivedfrom the list of differentially expressed genes using Ingenuity PathwayAnalysis (IPA) with microarray gene expression data from control andHMGA1 knock-down in MDA-MB-231 cells. From among 63 differentiallyexpressed genes as the focus gene set, the highest-scoring network wasEmbryonic Development, Tissue Development, and Cellular Development(score=69). Red nodes indicate up-regulation; green nodes indicatedown-regulation. Arrows and lines denote interactions between specificgenes within the network. A, activation; E, expression regulation; I,inhibition; L, proteolysis; LO, localization; M, biochemicalmodification; MB, membership of a group or complex; P, phosphorylation;PD, protein-DNA interaction; PP, protein-protein interaction; PR,protein-RNA interaction; RB, regulation of binding; RE, reaction; T,transcription; TR, translocation;

FIG. 5A and FIG. 5B show that silencing HMGA1 in MDA-MB-231 blocks theformation of foci to the lung following tail vein injections: FIG. 5Adepicts lung foci enumerated 3 weeks following tail vein injections ofcontrol or shHMGA1 MDA-MB-231 cells (n=3 for control mice; n=4 forshHMGA1 mice); and FIG. 5B is a graph of the mean number of tumorfoci±standard deviation, which shows a striking decrease in focifollowing injection of shHMGA1 MDA-MB-231 cells as compared to controls(p=0.007);

FIG. 6A, FIG. 6B, FIG. 6C and FIG. 6D show that silencing HMGA1 inMDA-MB-231 results in significant repression in HMGA1 mRNA and protein,with alterations in gene expression: FIG. 6A demonstrates thatindependent replicate experiments of MDA-MB-231 cells with or withoutHMGA1 knock-down result in silencing HMGA1 at the level of mRNA; FIG. 6Bdemonstrates that HMGA1 protein is also repressed following treatmentwith siRNA; FIG. 6C is a validation of genes in the HMGA1 signaturewhich shows that gene expression assessed by quantitative RT-PCR(qRT-PCR) parallels that of the microarray results; and FIG. 6D is atable comparing differential expression of the HMGA1 signatureidentified by microarray and qRT-PCR;

FIG. 7 shows the HMGA1 network derived from differentially expressedgenes in MDA-MB-231 with or without HMGA1 knock-down. From among 63differentially expressed genes as the focus gene set, the secondhighest-scoring network was Cardiovascular Disease, Cell Death andSurvival, and Nervous System Development and Function (score=46).Colors, arrows, lines and abbreviations are described under FIG. 4B.NF-KB, ERK, and MAPK are major nodes, which have been identified inprior studies of global gene expression profiles mediated by HMGA1(Schuldenfrei et al. (2011) BMC Genomics 12:549);

FIG. 8 demonstrates the oncogenic pathways activated by HMGA;

FIG. 9 demonstrates the targeting of HMGA1 in cancer stem cells;

FIG. 10A, FIG. 10B, and FIG. 10C demonstrate that HMGA1 correlates withpoor differentiation and decreased survival in pancreatic cancer; FIG.10A shows a well-differentiated tumor with low levels of HMGA1immunoreactivity; FIG. 10B shows a high-grade, poorly differentiatedtumor with high immunoreactivity. Patient survival (FIG. 10C) isdecreased with high levels of HMGA1;

FIG. 11 demonstrates HMGA1 expression levels in different pancreaticductal adenocarcinoma cell (PDAC) lines; gene expression was assessedvia quantitative RT-PCR (qRT-PCR);

FIG. 12A and FIG. 12B demonstrate that HMGA1 is silenced by shorthairpin RNA as seen by expression levels (via qRT-PCR) of HMGA1 with(red) and without (blue) shHMGA1; FIG. 12B shows protein levels byWestern blot;

FIG. 13 demonstrates that silencing HMGA1 via shHMGA disrupts cellproliferation in three PDAC cell lines (red), including twopatient-derived cell lines, as compared to the control (blue);

FIG. 14A and FIG. 14B demonstrate that silencing HMGA1 alters morphologyof pancreatic ductal adenocarcinoma cells. FIG. 14A shows morphology ofcontrol cells not treated with the HMGA1 shRNA (spindle-shaped,mesenchymal cells) as compared to the morphology of pancreatic ductaladenocarcinoma cells treated with shRNA targeting HMGA1 (rounded, morecuboidal-shaped cells; FIG. 14B);

FIG. 15A, FIG. 15B and FIG. 15C demonstrate that silencing HMGA1 viashHMGA blocks 3D sphere formation (FIG. 15A), PDAC colony formation(FIG. 1B), and migration and invasion (FIG. 15C);

FIG. 16A, FIG. 16B, and FIG. 16C demonstrate that silencing is cytotoxicin acute myeloid leukemia (AML) cell lines and that silencing incolorectal cancer cells halts proliferation and alters cell morphology.FIG. 16A shows silencing of HMGA1 in three AML cell lines and FIG. 16Bshows marked cytotoxicity in HMGA1 knockdown cells. FIG. 16C shows thatsilencing HMGA1 in colorectal cancer cells halts proliferation andalters cell morphology;

FIG. 17 demonstrates nanoparticle shRNA treatment in PDAC cell line 10.7xenografts. The PDAC cell line was injected into mice on tumors allowedto grow. By day 30, mice were treated with nanoparticles to delivershRNA to the tumors; and

FIG. 18A, FIG. 18B, and FIG. 18C demonstrate vacuolated cytoplasm ofmost tumor cells, individual cell death (arrows) and area of necrosis(outlined) in a shHMGA/treated tumor.

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

DETAILED DESCRIPTION

The presently disclosed subject matter now will be described more fullyhereinafter with reference to the accompanying Figures, in which some,but not all embodiments of the presently disclosed subject matter areshown. Like numbers refer to like elements throughout. The presentlydisclosed subject matter may be embodied in many different forms andshould not be construed as limited to the embodiments set forth herein;rather, these embodiments are provided so that this disclosure willsatisfy applicable legal requirements. Indeed, many modifications andother embodiments of the presently disclosed subject matter set forthherein will come to mind to one skilled in the art to which thepresently disclosed subject matter pertains having the benefit of theteachings presented in the foregoing descriptions and the associatedFigures. Therefore, it is to be understood that the presently disclosedsubject matter is not to be limited to the specific embodimentsdisclosed and that modifications and other embodiments are intended tobe included within the scope of the appended claims.

Emerging evidence suggests that tumor cells metastasize by co-optingstem cell transcriptional networks, although the molecular underpinningsof this process are poorly understood. Recent studies have identifiedthe high mobility group A1 (HMGA1) oncogene (Entrez Gene ID: 3159) as akey factor enriched in embryonic stem cells, adult stem cells, andrefractory or high-grade/poorly differentiated tumors (Ben-Porath et al.(2008) Nat. Genet. 40:499-507; Resar (2010) Cancer Research 70:436-439;Chou et al. (2011) Cell Res. 21:518-529; Zhou et al. (2001) Proc. Natl.Acad. Sci. USA 98:13966-13971; Karp et al. (2011) Blood 117:3302-3310;Nelson et al. (2011) Leuk Lymphoma 52:1999-2006; Schuldenfrei et al.(2011) BMC Genomics 12:549; Belton et al. (2012) PloS One 7:e30034; Shahand Resar (2012) Histol. Histopathol. 27:567-579; Wood et al. (2000)Mol. Cell. Biol. 20:5490-5502; Pedulla et al. (2001) Gene 271:51-58;Reeves and Beckerbauer (2001) Biochim. Biophys. Acta 1519:13-29; Reeveset al. (2001) Mol. Cell. Biol. 21:575-594; Dolde et al. (2002) BreastCancer Research and Treatment 71:181-191; Dhar et al. (2004) Oncogene23:4466-4476; Takaha et al. (2004) The Prostate 60:160-167; Hommura etal. (2004) Mol. Cancer Res. 2:305-314; Xu et al. (2004) Cancer Res.64:3371-3375; Tesfaye et al. (2007) Cancer Res. 67:3998-4004; Fusco andFedele (2007) Nat. Rev. Cancer 7:899-910; Di Cello et al. (2008)Molecular Cancer Therapuetics 7:2090-2095; Hillion et al. (2008) CancerRes. 68:10121-10127; Hillion et al. (2009) Mol. Cancer Res. 7:1803-1812;Hristov et al. (2010) Mod. Pathol. 23: 98-104; Reeves (2010) Biochim.Biophys. Acta 1799:3-14; Di Cello et al. (2013) Leuk. Lymphoma54:1762-1768; Williams et al. (2013) Anal. Bioanal. Chem. 405:5013-5030;Pomeroy et al. (2002) Nature 415: 436-442; Flohr et al. (2003) Histol.Histopathol. 18: 999-1004; Shah et al. (2012) PLoS One 7: e48533).

The HMGA1 gene encodes the HMGA1a and HMGA1b chromatin remodelingproteins, which result from alternatively spliced messenger RNA (Resar(2010) Cancer Research 70:436-439; Reeves and Beckerbauer (2001)Biochim. Biophys. Acta 1519:13-29; Fusco and Fedele (2007) Nat. Rev.Cancer 7:899-910; Reeves (2010) Biochim. Biophys. Acta 1799:3-14). Theselow molecular weight (thus high mobility group) protein isoforms bind tothe minor groove of chromatin at AT-rich regions. HMGA1 proteinsmodulate gene expression by altering chromatin structure andorchestrating the assembly of transcription factor complexes toenhanceosomes within enhancer or promoter regions throughout the genome.These proteins are highly expressed during embryogenesis with low orabsent levels in adult tissues.

HMGA1 is overexpressed in all aggressive cancers studied to date, andhigh levels portend a poor prognosis in diverse tumors (Ben-Porath etal. (2008) Nat. Genet. 40:499-507; Resar (2010) Cancer Research70:436-439; Chou et al. (2011) Cell Res. 21:518-529; Zhou et al. (2001)Proc. Natl. Acad. Sci. USA 98:13966-13971; Karp et al. (2011) Blood117:3302-3310; Nelson et al. (2011) Leuk Lymphoma 52:1999-2006;Schuldenfrei et al. (2011) BMC Genomics 12:549; Belton et al. (2012)PloS One 7:e30034; Shah and Resar (2012) Histol. Histopathol.27:567-579; Wood et al. (2000) Mol. Cell. Biol. 20:5490-5502; Pedulla etal. (2001) Gene 271:51-58; Reeves and Beckerbauer (2001) Biochim.Biophys. Acta 1519:13-29; Reeves et al. (2001) Mol. Cell. Biol.21:575-594; Dolde et al. (2002) Breast Cancer Research and Treatment71:181-191; Dhar et al. (2004) Oncogene 23:4466-4476; Takaha et al.(2004) The Prostate 60:160-167; Hommura et al. (2004) Mol. Cancer Res.2:305-314; Xu et al. (2004) Cancer Res. 64:3371-3375; Tesfaye et al.(2007) Cancer Res. 67:3998-4004; Fusco and Fedele (2007) Nat. Rev.Cancer 7:899-910; Di Cello et al. (2008) Molecular Cancer Therapuetics7:2090-2095; Hillion et al. (2008) Cancer Res. 68:10121-10127; Hillionet al. (2009) Mol. Cancer Res. 7:1803-1812; Hristov et al. (2010) Mod.Pathol. 23: 98-104; Reeves (2010) Biochim. Biophys. Acta 1799:3-14; DiCello et al. (2013) Leuk. Lymphoma 54:1762-1768; Williams et al. (2013)Anal. Bioanal. Chem. 405:5013-5030; Pomeroy et al. (2002) Nature 415:436-442; Flohr et al. (2003) Histol. Histopathol. 18: 999-1004). Infact, HMGA1 proteins are the most abundant nonhistone chromatin bindingproteins found in cancer cells. A recent landmark paper demonstratedthat HMGA1 is essential for the cellular reprogramming of somatic cellsto induced pluripotent stem cells by the four Yamanaka factors (Oct4,Sox2, Klf4, cMyc) (Shah et al. (2012) PLoS One 7: e48533). HMGA1 inducesexpression of key stem cell transcriptional networks in normal embryonicstem cells and during cellular reprogramming The presently disclosedsubject matter relates in part to the discovery that HMGA1 is a centralfactor in reprogramming cancer stem cells. In particular, it wasdiscovered that the HMGA1 gene drives metastatic progression intriple-negative breast cancer cells (MDA-MB-231, Hs578T) byreprogramming cancer cells to a stem-like state. Silencing HMGA1expression in invasive, aggressive breast cancer cells dramaticallyhalted cell growth and resulted in striking morphologic changes frommesenchymal-like, spindle-shaped cells to cuboidal, epithelial-likecells. Mesenchymal genes (Vimentin, Twist) were repressed, whileE-cadherin was induced in knock-down cells. Silencing HMGA1 also blockedoncogenic properties, including proliferation, migration, invasion, andorthotopic tumorigenesis. Metastatic progression following mammaryimplantation was almost completely abrogated in the HMGA1 knock-downcells. Moreover, silencing HMGA1 inhibited the stem cell properties anddepleted breast cancer initiator/cancer stem cells. An HMGA1 signaturein triple-negative breast cancer cells was also discovered that washighly enriched in embryonic stem cells. Accordingly, in someembodiments, the presently disclosed subject matter provides methods forinhibiting cancer stem cells and growth of aggressive and/or poorlydifferentiated metastatic tumors, by inhibiting the expression of thehigh mobility group A1 (HMGA1) gene.

I. Methods of Inhibiting Cancer Stem Cells

It has been found that silencing HMGA1 reprograms aggressive stem-likecancer cells into non stem-like cells with slow growth and alteredproperties. Accordingly, in some embodiments, the presently disclosedsubject matter provides a method of inhibiting at least one cancer stemcell, the method comprising contacting at least one cancer stem cellwith an effective amount of at least one HMGA1 inhibitor. Cancer stemcells (CSCs) are cancer cells that possess characteristics associatedwith normal stem cells, specifically the ability to differentiate intomultiple cell types. CSCs may generate tumors through the stem cellprocesses of self-renewal and differentiation and are proposed topersist in tumors as a distinct population. In addition, they appear tobe highly drug-resistant cells.

As used herein, an HMGA1 inhibitor is an agent that inhibits target geneexpression (i.e., HMGA1 gene expression). As used herein, “inhibition oftarget gene expression” includes any decrease in expression or proteinactivity or level of the target gene (HMGA1 gene) or protein encoded bythe target gene (HMGA1 protein). The decrease may be of at least 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more as comparedto the expression of a target gene or the activity or level of theprotein encoded by a target gene which has not been targeted by an RNAinterfering agent. Certain exemplary methods of assaying for HMGA1 geneexpression or HGMA1 protein activity include, but are not limited to,those methods disclosed herein as well as assays known to those skilledin the art (see, e.g., Liau et al. (2006) Cancer Res. 66:11613-11622;Liu et al. (2012) Biotechnol. Appl. Biochem. 59:1-5).

HMGA1 inhibitors for use in the presently disclosed methods include RNAinterfering agents. An “RNA interfering agent” as used herein, isdefined as any agent which interferes with or inhibits expression of atarget gene, e.g., a marker of the presently disclosed subject matter,by RNA interference (RNAi). Such RNA interfering agents include, but arenot limited to, nucleic acid molecules including RNA molecules which arehomologous to the target gene, e.g., a marker of the presently disclosedsubject matter, or a fragment thereof, short interfering RNA (siRNA),and small molecules which interfere with or inhibit expression of atarget gene by RNA interference (RNAi). In some embodiments, at leastone HMG1 inhibitor is an RNA interfering agent.

“RNA interference (RNAi)” is an evolutionally conserved process wherebythe expression or introduction of RNA of a sequence that is identical orhighly similar to a target gene results in the sequence specificdegradation or specific post-transcriptional gene silencing (PTGS) ofmessenger RNA (mRNA) transcribed from that targeted gene (see Coburn &Cullen (2002) J. Virol. 76:9225), thereby inhibiting expression of thetarget gene (see, e.g., U.S. Patent Application Nos: 20030153519A1;20030167490A1; and U.S. Pat. Nos. 6,506,559; 6,573,099). In oneembodiment, the RNA is double stranded RNA (dsRNA). This process hasbeen described in plants, invertebrates, and mammalian cells. In nature,RNAi is initiated by the dsRNA-specific endonuclease Dicer, whichpromotes processive cleavage of long dsRNA into double-strandedfragments termed siRNAs. siRNAs are incorporated into a protein complexthat recognizes and cleaves target mRNAs. RNAi can also be initiated byintroducing nucleic acid molecules, e.g., synthetic siRNAs or RNAinterfering agents, to inhibit or silence the expression of targetgenes.

The presently disclosed subject matter also contemplates “shortinterfering RNA” (siRNA), also referred to herein as “small interferingRNA.” Such a molecule is defined as an agent which functions to inhibitexpression of a target gene, e.g., by RNAi. As used herein, the termsiRNA is intended to be equivalent to any term in the art defined as amolecule capable of mediating sequence-specific RNAi. Such equivalentsinclude, for example, double-stranded RNA (dsRNA), microRNA (mRNA),short hairpin RNA (shRNA), short interfering oligonucleotide, andpost-transcriptional gene silencing RNA (ptgsRNA). An siRNA may bechemically synthesized, may be produced by in vitro transcription, ormay be produced within a host cell. In one embodiment, siRNA is a doublestranded RNA (dsRNA) molecule of about 15 to about 40 nucleotides inlength, preferably about 15 to about 28 nucleotides, more preferablyabout 19 to about 25 nucleotides in length, and more preferably about19, 20, 21, or 22 nucleotides in length, and may contain a 3′ and/or 5′overhang on each strand having a length of about 0, 1, 2, 3, 4, or 5nucleotides. The length of the overhang is independent between the twostrands, i.e., the length of the overhang on one strand is not dependenton the length of the overhang on the second strand. Preferably the siRNAis capable of promoting RNA interference through degradation or specificpost-transcriptional gene silencing (PTGS) of the target messenger RNA(mRNA).

In another embodiment, an siRNA is a small hairpin (also called stemloop) RNA (shRNA). In one embodiment, these shRNAs are composed of ashort (e.g., 19-25 nucleotide) antisense strand, followed by a 5-9nucleotide loop, and the analogous sense strand. Alternatively, thesense strand may precede the nucleotide loop structure and the antisensestrand may follow. These shRNAs may be contained in plasmids,retroviruses, and lentiviruses and expressed from, for example, the polIII U6 promoter, or another promoter (see, e.g., Stewart et al. (2003)RNA 9:493-501). In a particular embodiment, the siRNA is an shRNA thattargets the nucleotide sequence of SEQ ID NO:1 (see, e.g., Liau et al.(2006) Cancer Res. 66:11613-11622).

As used herein, inhibition of at least one cancer stem cell includes,but is not limited to, inhibition of oncogenic properties associatedwith both tumor initiation (orthotopic tumorigenesis) and tumorprogression (migration, invasion, and metastatic progression), forexample, inhibition of growth of cancer stem cells as compared to thegrowth of untreated or mock treated cells, inhibition of metastases,induction of cancer cell senescence, induction of cancer cell death, andreduction of tumor size.

As used herein, the term “contacting” means any action that results inat least one HMGA1 inhibitor of the presently disclosed subject matterphysically contacting at least one cell, such as a cancer stem cell. Itthus may comprise exposing the cell(s) to the HMGA1 inhibitor in anamount sufficient to result in contact of at least one HMGA1 inhibitorwith at least one cell. The method can be practiced in vitro or ex vivoby introducing, and preferably mixing, the HMGA1 inhibitor and cells ina controlled environment, such as a culture dish or tube. The method canbe practiced in vivo, in which case contacting means exposing at leastone cell in a subject to at least one HMGA1 inhibitor of the presentlydisclosed subject matter, such as administering the HMGA1 inhibitor to asubject via any suitable route. According to the presently disclosedsubject matter, contacting may comprise introducing, exposing, and thelike, the HMGA1 inhibitor at a site distant to the cells to becontacted, and allowing the bodily functions of the subject, or natural(e.g., diffusion) or man-induced (e.g., swirling) movements of fluids toresult in contact of the HMGA1 inhibitor and cell(s). In general, theterm “effective amount” refers to the amount of an agent, such as anHMGA1 inhibitor, to elicit the desired biological response, such asinhibition of a cancer stem cell.

In addition, “a therapeutically effective amount,” of a therapeuticagent refers to the amount of the agent necessary to elicit the desiredbiological response. As will be appreciated by those of ordinary skillin this art, the effective amount of an agent may vary depending on suchfactors as the desired biological endpoint, the agent to be delivered,the composition of the pharmaceutical composition, the target tissue orcell, and the like. More particularly, the term “effective amount”refers to an amount sufficient to produce the desired effect, e.g., toreduce or ameliorate the severity, duration, progression, or onset of adisease, disorder, or condition (e.g., aggressive and/or poorlydifferentiated metastatic cancer), or one or more symptoms thereof;prevent the advancement of a disease, disorder, or condition, cause theregression of a disease, disorder, or condition; prevent the recurrence,development, onset or progression of a symptom associated with adisease, disorder, or condition, or enhance or improve the prophylacticor therapeutic effect(s) of another therapy. Accordingly, as usedherein, treatment of aggressive and/or poorly differentiated metastaticcancer, includes, but is not limited to, reduction in cancer growth ortumor burden, induction of cancer cell senescence, induction ofapoptosis of cancer cells, induction of cancer cell death, inhibition ofangiogenesis, enhancement of cancer cell apoptosis, and inhibition ofmetastases.

In some embodiments, inhibiting at least one cancer stem cell isselected from the group consisting of i) inhibiting proliferation of theat least one cancer stem cell; ii) inhibiting self-renewal of the atleast one cancer stem cell; iii) inhibiting anchorage-independent growthof the at least one cancer stem cell; iv) inhibiting migration of the atleast one cancer stem cell; v) inhibiting invasion of the at least onecancer stem cell; vi) reprogramming the at least one cancer stem cellfrom a stem-like state that is refractory to apoptosis to a nonstem-like state that is susceptible to apoptosis; and vii) combinationsthereof As used herein, the term “proliferation” refers to an increasein the number of cells as a result of cell growth and cell division.Thus, inhibiting proliferation of a cancer stem cell means to reduce itsability to undergo cell growth and/or cell division. “Self-renewal”refers to the process by which stem cells divide to make more stemcells, thereby perpetuating the stem cell pool. Self-renewal is divisionwith maintenance of the undifferentiated state. Some cancer arises frommutations that inappropriately activate self-renewal programs.“Migration” refers to the ability of a cell to move. “Invasion” refersto the ability of cells to become motile and to navigate through theextracellular matrix within a tissue or to infiltrate neighboringtissues. Cancer cells that become invasive may disseminate to secondarysites and form metastases. “Anchorage-independent growth” refers to theability of a cell to have colony forming capacity in semisolid media,which is connected with tumor cell aggressiveness in vivo.“Reprogramming” a cell refers to causing a change in the cell, forexample, by changing a cancer stem cell from a cell that is refractoryor resistant to apoptosis (e.g., as a result of exposure to achemotherapeutic agent) to a state where the cell is susceptible toapoptosis. As used herein, “apoptosis”, also referred to as programmedcell death, refers to the process of cell self-destruction.

In some embodiments, at least one cancer stem cell is in an aggressiveand/or poorly differentiated metastatic tumor, and inhibiting the atleast one cancer stem cell inhibits at least one of: i) growth of theaggressive and/or poorly differentiated metastatic tumor; ii)proliferation of the aggressive and/or poorly differentiated metastatictumor; iii) migration of the aggressive and/or poorly differentiatedmetastatic tumor; iv) invasion of the aggressive and/or poorlydifferentiated metastatic tumor; v) initiation of new aggressive and/orpoorly differentiated metastatic tumors; and vi) combinations thereof Asused herein, the term “aggressive” in the context of a tumor/cancermeans that the tumor/cancer exhibits rapid growth, is more likely tohave spread by the time it has been diagnosed, and is more refractorythan other non-aggressive forms of the tumor/cancer in that it is morelikely to recur after treatment as compared to the non-aggressive formof the tumor/cancer. As used herein, the term “poorly differentiated”refers to a cell that is abnormal looking as compared to a normal cell.Poorly differentiated cells in a tumor are an indicator of a moreaggressive tumor. A “metastatic tumor” or “metastatic cancer” is a tumoror cancer that has spread from the place where it first started toanother place in the body.

HMGA1 is overexpressed in bulk tumor mass of aggressive and/or poorlydifferentiated metastatic cancers with the highest levels of HMGA1overexpression occurring in the cancer stem cells of the aggressiveand/or poorly differentiated metastatic cancer. Accordingly, in someembodiments, at least one cancer stem cell expresses greater levels ofHMGA1 as compared to non-stem cancer cells in the aggressive and/orpoorly differentiated metastatic tumor. In some embodiments, at leastone cancer stem cell expresses greater levels of HMGA1 as compared tonormal tissue or precursor lesions. In some embodiments, an aggressiveand/or poorly differentiated metastatic tumor expresses greater levelsof HMGA1 as compared to normal tissue or precursor lesions. As usedherein, the term “greater levels” means a level of HMGA1 in a samplethat is higher than the level of expression of HMGA1 in a control sampleby at least 1.5 fold, 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 2.0 fold,2.1 fold, 2.2 fold, 2.3 fold, 2.4 fold, 2.5 fold, 2.6 fold, 2.7 fold,2.8 fold, 2.9 fold, 3.0 fold, 3.1 fold, 3.2 fold, 3.3 fold, 3.4 fold,3.5 fold, 3.6 fold, 3.7 fold, 3.8 fold, 3.9 fold, 4.0 fold, 4.1 fold,4.2 fold, 4.3 fold, 4.4 fold, 4.5 fold, 4.6 fold, 4.7 fold, 4.8 fold,4.9 fold, 5.0 fold or more. In other embodiments, the term “greaterlevels” means a level of HMGA1 in a sample that is higher than the levelof HMGA1 in a control sample by at least 10 fold, 20 fold, 50 fold, 100fold, 200 fold, 300 fold, 400 fold or more.

A “cancer” in a patient refers to the presence of cells possessingcharacteristics typical of cancer-causing cells, for example,uncontrolled proliferation, loss of specialized functions, immortality,significant metastatic potential, significant increase in anti-apoptoticactivity, rapid growth and proliferation rate, and certaincharacteristic morphology and cellular markers. In some circumstances,cancer cells will be in the form of a tumor; such cells may existlocally within an animal, or circulate in the blood stream asindependent cells. Cancer as used herein includes newly diagnosed orrecurrent cancers, including without limitation, blastomas, carcinomas,gliomas, leukemias, lymphomas, melanomas, myeloma, and sarcomas. Canceras used herein includes, but is not limited to, head cancer, neckcancer, head and neck cancer, lung cancer, breast cancer, prostatecancer, colorectal cancer, esophageal cancer, stomach cancer,leukemia/lymphoma, uterine cancer, skin cancer, endocrine cancer,urinary cancer, pancreatic cancer, gastrointestinal cancer, ovariancancer, cervical cancer, and adenomas. In some embodiments, the cancercomprises Stage 0 cancer. In some embodiments, the cancer comprisesStage I cancer. In some embodiments, the cancer comprises Stage IIcancer. In some embodiments, the cancer comprises Stage III cancer. Insome embodiments, the cancer comprises Stage IV cancer. In someembodiments, the cancer is refractory and/or metastatic. In someembodiments, at least one cancer stem cell is selected from the groupconsisting of a triple-negative breast cancer cell, a pancreatic ductaladenocarcinoma cell, a colorectal cancer cell, and a leukemia cell.

In some embodiments, at least one HMGA1 inhibitor reduces the expressionlevel and/or activity of HMGA1. As used herein, the term “expressionlevel” refers to the amount of a mRNA or protein detected. Levels can bedetected at the transcriptional level, the translational level, and thepost-translational level, for example. “mRNA expression levels” refersto the amount of mRNA detected in a sample and “protein expressionlevels” refers to the amount of protein detected in a sample.

In some embodiments, the methods further comprise contacting at leastone cancer stem cell with a chemotherapeutic agent. A “chemotherapeuticagent” is used to connote a compound or composition that is administeredin the treatment of cancer. Chemotherapeutic agents useful in methods,compositions, and kits disclosed herein include, but are not limited to,alkylating agents such as thiotepa, temozolomide, and cyclophosphamide;alkyl sulfonates such as busulfan, improsulfan and piposulfan;aziridines such as benzodopa, carboquone, meturedopa, and uredopa;ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamime; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, ranimustine;antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine,bleomycins, cactinomycin, calicheamicin, carabicin, caminomycin,carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytosine arabinoside, dideoxyuridine, doxifluridine, enocitabine,floxuridine, 5-FU; androgens such as calusterone, dromostanolonepropionate, epitiostanol, mepitiostane, testolactone; anti-adrenals suchas aminoglutethimide, mitotane, trilostane; folic acid replenishers suchas folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone;mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK; razoxane;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (Ara-C); taxoids, e.g. paclitaxel and docetaxel;chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; platinumanalogs such as cisplatin and carboplatin; vinblastine; platinum;etoposide; ifosfamide; mitomycin C; mitoxantrone; vincristine;vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin;xeloda; ibandronate; CPT11; topoisomerase inhibitor RFS 2000;difluoromethylornithine; retinoic acid; esperamicins; capecitabine;immune system blockers, e.g. rapamycin; amino acid modifiers, e.g.asparaginase; and pharmaceutically acceptable salts, acids orderivatives of any of the above. Chemotherapeutic agents also includeanti-hormonal agents that act to regulate or inhibit hormone action ontumors such as anti-estrogens including for example tamoxifen,raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen,trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston);and anti-androgens such as flutamide, nilutamide, bicalutamide,leuprolide, and goserelin; and pharmaceutically acceptable salts, acidsor derivatives of any of the above.

In some embodiments, the chemotherapeutic agent is a topoisomeraseinhibitor. Topoisomerase inhibitors are chemotherapy agents thatinterfere with the action of a topoisomerase enzyme (e.g., topoisomeraseI or II). Topoisomerase inhibitors include, but are not limited to,doxorubicin HCl, daunorubicin citrate, mitoxantrone HCl, actinomycin D,etoposide, topotecan HCl, teniposide, and irinotecan, as well aspharmaceutically acceptable salts, acids, or derivatives of any ofthese.

In some embodiments, the chemotherapeutic agent is an anti-metabolite.An anti-metabolite is a chemical with a structure that is similar to ametabolite required for normal biochemical reactions, yet differentenough to interfere with one or more normal functions of cells, such ascell division. Anti-metabolites include, but are not limited to,gemcitabine, fluorouracil, capecitabine, methotrexate sodium,ralitrexed, pemetrexed, tegafur, cytosine arabinoside, thioguanine,5-azacytidine, 6-mercaptopurine, azathioprine, 6-thioguanine,pentostatin, fludarabine phosphate, and cladribine, as well aspharmaceutically acceptable salts, acids, or derivatives of any ofthese.

In certain embodiments, the chemotherapeutic agent is an antimitoticagent, including, but not limited to, agents that bind tubulin. In someembodiments, the agent is a taxane. In certain embodiments, the agent ispaclitaxel or docetaxel, or a pharmaceutically acceptable salt, acid, orderivative of paclitaxel or docetaxel. In certain alternativeembodiments, the antimitotic agent comprises a vinca alkaloid, such asvincristine, binblastine, vinorelbine, or vindesine, or pharmaceuticallyacceptable salts, acids, or derivatives thereof. In some embodiments,the chemotherapeutic agent is gemcitabine.

In some embodiments, at least one cancer stem cell is contacted in asubject. In some embodiments, the subject is a human subject.

II. Methods of Treating Aggressive and/or Poorly DifferentiatedMetastatic Cancer

In some embodiments, an aggressive and/or poorly differentiatedmetastatic cancer cell may be contacted with an HGMA1 inhibitor within asubject, and such contact may result in treatment of aggressive and/orpoorly differentiated metastatic cancer in the subject. In someembodiments, at least some of the cells from the aggressive and/orpoorly differentiated metastatic cancer comprise cancer stem cells.Accordingly, the presently disclosed subject matter also providesmethods of treating aggressive and/or poorly differentiated metastaticcancer in a subject in need thereof In such embodiments, the methodsinclude administering a therapeutically effective amount of at least oneHMGA1 inhibitor to a subject in need thereof to treat aggressive and/orpoorly differentiated metastatic cancer. In some embodiments, theaggressive and/or poorly differentiated metastatic cancer is selectedfrom the group consisting of triple-negative breast cancer, pancreaticductal adenocarcinoma cell, colorectal cancer cell, and leukemia. Insome embodiments, the aggressive and/or poorly differentiated metastaticcancer comprises at least one cancer stem cell that overexpresses HMGA1protein.

The presently disclosed methods may further comprise selecting thesubject for treatment of the aggressive and/or poorly differentiatedmetastatic cancer with at least one HMGA1 inhibitor. In someembodiments, selecting the subject for treatment of the aggressiveand/or poorly differentiated metastatic cancer comprises: (i) obtaininga biological sample comprising cells from the aggressive and/or poorlydifferentiated metastatic cancer; (ii) assaying the level of HMGA1expression in the cells from the aggressive and/or poorly differentiatedmetastatic cancer; (iii) comparing the level of HMGA1 expression in thecells to the level of HMGA1 expression in a normal control cell; and(iv) selecting the subject for treatment of the aggressive and/or poorlydifferentiated metastatic cancer with the at least one HMGA1 inhibitorif the level of HMGA1 expression in the cells is greater than the levelof HMGA1 expression in the normal control cell.

The terms “sample,” “subject sample,” “biological sample,” and the like,encompass a variety of sample types obtained from a subject, individual,or subject and can be used in a diagnostic or monitoring assay. Thesubject sample may be obtained from a healthy subject, a diseasedsubject or a subject having associated symptoms of cancer. Moreover, asample obtained from a subject can be divided and only a portion may beused for diagnosis. Further, the sample, or a portion thereof, can bestored under conditions to maintain sample for later analysis. Thedefinition specifically encompasses blood and other liquid samples ofbiological origin (including, but not limited to, peripheral blood,serum, plasma, cerebrospinal fluid, urine, saliva, stool and synovialfluid), solid tissue samples such as a biopsy specimen or tissuecultures or cells derived therefrom and the progeny thereof In aspecific embodiment, a sample comprises a cancer tissue sample. Thedefinition also includes samples that have been manipulated in any wayafter their procurement, such as by centrifugation, filtration,precipitation, dialysis, chromatography, treatment with reagents,washed, or enriched for certain cell populations. The terms furtherencompass a clinical sample, and also include cells in culture, cellsupernatants, tissue samples, organs, and the like. Samples may alsocomprise fresh-frozen and/or formalin-fixed, paraffin-embedded tissueblocks, such as blocks prepared from clinical or pathological biopsies,prepared for pathological analysis or study by immunohistochemistry. Insome embodiments, the biological sample is selected from the groupconsisting of a breast tissue sample, a pancreatic tissue sample, acolon tissue sample, and a bone marrow tissue sample.

In some embodiments, at least one HMGA1 inhibitor is formulated fordelivery in a nanoparticle. As used herein, the term “nanoparticle,”refers to a particle having at least one dimension in the range of about1 nm to about 1000 nm, including any integer value between 1 nm and 1000nm (including about 1, 2, 5, 10, 20, 50, 60, 70, 80, 90, 100, 200, 500,and 1000 nm and all integers and fractional integers in between).

As used herein, the term “subject” treated by the presently disclosedmethods in their many embodiments is desirably a human subject, althoughit is to be understood that the methods described herein are effectivewith respect to all vertebrate species, which are intended to beincluded in the term “subject.” Accordingly, a “subject” can include ahuman subject for medical purposes, such as for the diagnosis ortreatment of an existing disease, disorder, condition or theprophylactic diagnosis or treatment for preventing the onset of adisease, disorder, or condition or an animal subject for medical,veterinary purposes, or developmental purposes. Suitable animal subjectsinclude mammals including, but not limited to, primates, e.g., humans,monkeys, apes, gibbons, chimpanzees, orangutans, macaques and the like;bovines, e.g., cattle, oxen, and the like; ovines, e.g., sheep and thelike; caprines, e.g., goats and the like; porcines, e.g., pigs, hogs,and the like; equines, e.g., horses, donkeys, zebras, and the like;felines, including wild and domestic cats; canines, including dogs;lagomorphs, including rabbits, hares, and the like; and rodents,including mice, rats, guinea pigs, and the like. An animal may be atransgenic animal In some embodiments, the subject is a human including,but not limited to, fetal, neonatal, infant, juvenile, and adultsubjects. Further, a “subject” can include a patient afflicted with orsuspected of being afflicted with a disease, disorder, or condition.Thus, the terms “subject” and “patient” are used interchangeably herein.Subjects also include animal disease models (e.g., rats or mice used inexperiments, and the like).

As described herein, the presently disclosed HMGA1 inhibitor can beadministered to a subject for therapy by any suitable route ofadministration, including orally, nasally, transmucosally, ocularly,rectally, intravaginally, parenterally, including intramuscular,subcutaneous, intramedullary injections, as well as intrathecal, directintraventricular, intravenous, intra-articular, intra-sternal,intra-synovial, intra-hepatic, intralesional, intracranial,intraperitoneal, intranasal, or intraocular injections,intracisternally, topically, as by powders, ointments or drops(including eyedrops), including buccally and sublingually,transdermally, through an inhalation spray, or other modes of deliveryknown in the art.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of at least one HMGA1 inhibitor such thatit enters the patient's system and, thus, is subject to metabolism andother like processes, for example, subcutaneous administration.

The phrases “parenteral administration” and “administered parenterally”as used herein mean modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intarterial, intrathecal,intracapsular, intraorbital, intraocular, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal and intrasternalinjection and infusion.

Regardless of the route of administration selected, compositionscomprising an HMGA1 inhibitor may be formulated into pharmaceuticallyacceptable dosage forms. One skilled in the art can select appropriateformulation components, such as carriers, buffers, adjuvants, etc.,according to the route of administration and/or the subject beingtreated.

Actual dosage levels of an HMGA1 inhibitor can be varied so as to obtainan amount of the active ingredient that is effective to achieve thedesired therapeutic response for a particular subject, composition,route of administration, and disease, disorder, or condition withoutbeing toxic to the subject. The selected dosage level will depend on avariety of factors including the activity of the particular compositionemployed, the route of administration, the time of administration, therate of excretion of the particular composition being employed, theduration of the treatment, other drugs, and/or materials used incombination with the particular composition employed, the age, sex,weight, condition, general health and prior medical history of thepatient being treated, and like factors well known in the medical arts.Accordingly, a physician having ordinary skill in the art can readilydetermine and prescribe the effective amount of the presently disclosedcomposition required. Accordingly, the dosage range for administrationwill be adjusted by the physician as necessary, as described more fullyelsewhere herein.

III. Methods Of Selecting a Subject with Aggressive and/or PoorlyDifferentiated Metastatic Cancer

In some embodiments, the presently disclosed method is a method ofselecting a subject with an aggressive and/or poorly differentiatedmetastatic cancer for treatment with at least one HMGA1 inhibitor, themethod comprising: (a) obtaining a biological sample from the subject,wherein the biological sample comprises cells from the aggressive and/orpoorly differentiated metastatic cancer; (b) determining the level ofexpression of HMGA1 in the biological sample; (c) comparing the level ofexpression of HMGA1 in the biological sample with the level ofexpression of HMGA1 in a control sample; and (d) selecting the subjectwith aggressive and/or poorly differentiated metastatic cancer fortreatment with at least one HMGA1 inhibitor when the level of HMGA1expression in the biological sample is greater than the level of HMGA1expression in the control sample.

IV. Methods of Diagnosing an Aggressive and/or Poorly DifferentiatedMetastatic Cancer

The presently disclosed subject matter also provides methods ofdiagnosing an aggressive and/or poorly differentiated metastatic cancerin a subject in need thereof. In such embodiments, the methods comprise:(a) obtaining a biological sample from the subject, wherein thebiological sample comprises cells suspected of being aggressive and/orpoorly differentiated metastatic cancer cells; (b) determining the levelof expression of two or more genes selected from Table 1 in thebiological sample; and (c) comparing the level of expression of the twoor more genes in the biological sample with the level of expression ofthe two or more genes in a control sample; wherein a significantdifference in the level of expression of the two or more genes in thebiological sample compared to the level of expression of the two or moregenes in the control sample indicates that the biological samplecomprises at least one aggressive and/or poorly differentiatedmetastatic cancer cell.

TABLE 1 Differentially Expressed Genes Induced By HMGA1 Gene Entrez GeneID Direction SLC17A5 26503 Down Expressed PGRMC1 10857 Down ExpressedTMCO1 54499 Up Expressed SLC9A6 10479 Down Expressed ARL2BP 23568 DownExpressed DRAM2 128338 Down Expressed PLAT 5327 Down Expressed SEPT24735 Down Expressed PSG4 5672 Up Expressed RPS10P7 376693 Down ExpressedINPP5A 3632 Down Expressed CAMK2N1 55450 Down Expressed OR6X1 390260 UpExpressed TGFBR2 7048 Up Expressed SLC37A2 219855 Down Expressed GRB22885 Down Expressed B3GALT5 10317 Down Expressed TAF9B 51616 DownExpressed HIST1H4L 8368 Down Expressed FOS 2353 Up Expressed SRPX 8406Down Expressed LIPA 3988 Down Expressed HMGA1 3159 Down Expressed CNDP255748 Down Expressed BOK 666 Down Expressed ABCA1 19 Down ExpressedMSRB3 253827 Up Expressed WSB2 55884 Down Expressed CLIP1 6249 DownExpressed WIPF1 7456 Down Expressed MYEOV 26579 Down Expressed SLC26A21836 Up Expressed WDFY1 57590 Up Expressed SYPL1 6856 Down ExpressedSTEAP3 55240 Down Expressed C5orf28 64417 Up Expressed IRAK2 3656 DownExpressed NFIB 4781 Up Expressed LOC100134868 100134868 Up ExpressedIPMK 253430 Down Expressed ICK 22858 Down Expressed PORCN 64840 DownExpressed SFXN1 94081 Up Expressed APOL6 80830 Down Expressed C12orf4979794 Down Expressed PIGO 84720 Down Expressed AKAP6 9472 Up ExpressedULBP3 79465 Down Expressed GPR45 11250 Down Expressed RACGAP1 29127 DownExpressed MT1E 4493 Down Expressed C3orf34 84984 Up Expressed STX19415117 Down Expressed GRAMD3 65983 Up Expressed SLC1A3 6507 Up ExpressedHMGCS1 3157 Up Expressed SEC24D 9871 Up Expressed MAP3K13 9175 DownExpressed GPR110 266977 Down Expressed TSPAN11 441631 Down ExpressedOR6F1 343169 Down Expressed PLAU 5328 Down Expressed ALDH1A3 220 DownExpressed

As used herein, the term “biomarker” refers to any gene, RNA or proteinwhose level of expression in a cell or tissue is altered in some waycompared to that of a normal or healthy cell or tissue. Biomarkers ofthe presently disclosed subject matter are selective for an aggressiveand/or poorly differentiated metastatic cancer and comprise two or moregenes comprising embryonic development genes, tissue development genes,cellular development genes, cell death and survival genes, cell movementgenes, or a combination thereof In particular, biomarkers of thepresently disclosed subject matter that are selective for aggressiveand/or poorly differentiated metastatic cancer include 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62 or 63 genes selected from Table 1 in the biological sample.

In some embodiments, the significant difference in expression of two ormore genes in the biological sample compared to the control samplecomprises increased expression of TMCO1, PSG4, OR6X1, TGFBR2, FOS,MSRB3, SLC26A2, WDFY1, C5orf28, NFIB, LOC100134868, SFXN1, AKAP6,C3orf34, GRAMD3, SLC1A3, HMGCS1, and/or SEC24D in the biological sample.

In some embodiments, the significant difference in expression of two ormore genes in the biological sample compared to the control samplecomprises decreased expression of SLC17A5, PGRMC1, SLC9A6, ARL2BP,DRAM2, PLAT, SEPT2, RPS10P7, INPP5A, CAMK2N1, SLC37A2, GRB2, B3GALT5,TAF9B, HIST1H4L, SRPX, LIPA, HMGA1, CNDP2, BOK, ABCA1, WSB2, CLIP1,WIPF1, MYEOV, SYPL1, STEAP3, IRAK2, IPMK, ICK, PORCN, APOL6, C12orf49,PIGO, ULBP3, GPR45, RACGAP1, MT1E, STX19, MAP3K13, GPR110, TSPAN11,OR6F1, PLAU, and/or ALDH1A3 in the biological sample.

In another embodiment, the significant difference in expression of twoor more genes in the biological sample compared to the control samplecomprises increased expression of TGFβ1, FOS, cMYC, STAT3, and/or TMCO1in the biological sample and/or decreased expression of ARL2BP, PGRMC1,and/or GRB2 in the biological sample.

As used herein, the term “level of expression” of a biomarker refers tothe amount of biomarker detected. Levels of biomarker can be detected atthe transcriptional level, the translational level, and thepost-translational level, for example. As used herein, the term“diagnosing” refers to the process of attempting to determine oridentify a disease or disorder.

As used herein, the term “comparing” refers to making an assessment ofhow the proportion or level of one or more biomarkers in a sample from asubject relates to the proportion or level of the corresponding one ormore biomarkers in a standard or control sample. For example,“comparing” may refer to assessing whether the proportion or level ofone or more biomarkers in a sample from a subject is the same as, moreor less than, or different from the proportion or level of thecorresponding one or more biomarkers in a standard or control sample.More specifically, the term may refer to assessing whether theproportion or level of one or more biomarkers in a sample from a subjectis the same as, more or less than, different from or otherwisecorresponds (or not) to the proportion or level of predefined biomarkerlevels that correspond to, for example, a subject having an aggressiveand/or poorly differentiated metastatic cancer. In a specificembodiment, the term “comparing” refers to assessing whether the levelof one or more biomarkers of the presently disclosed subject matter in asample from a subject is the same as, more or less than, different fromor otherwise correspond (or not) to levels of the same biomarkers in acontrol sample (e.g., predefined levels that correlate to individualswithout an aggressive and/or poorly differentiated metastatic cancer,standard biomarker levels, and the like).

As used herein, the terms “indicates” or “correlates” (or “indicating”or “correlating,” or “indication” or “correlation,” depending on thecontext) in reference to a parameter, e.g., a modulated proportion orlevel in a sample from a subject, may mean that the subject has anaggressive and/or poorly differentiated metastatic cancer. In specificembodiments, the parameter may comprise the level of one or morebiomarkers of the presently disclosed subject matter. A particular setor pattern of the amounts of one or more biomarkers may indicate that asubject has an aggressive and/or poorly differentiated metastaticcancer. In other embodiments, a particular set or pattern of the amountsof one or more biomarkers may be correlated to a subject beingunaffected (i.e., indicates a subject does not have an aggressive and/orpoorly differentiated metastatic cancer). In certain embodiments,“indicating,” or “correlating,” as used according to the presentlydisclosed subject matter, may be by any linear or non-linear method ofquantifying the relationship between levels of biomarkers to a standard,control or comparative value for the assessment of the diagnosis,prediction of an aggressive and/or poorly differentiated metastaticcancer, assessment of efficacy of clinical treatment, identification ofa subject that may respond to a particular treatment regime orpharmaceutical agent, monitoring of the progress of treatment, and inthe context of a screening assay, for the identification of atherapeutic to treat an aggressive and/or poorly differentiatedmetastatic cancer.

A “subject” can include a subject afflicted with or suspected of beingafflicted with a condition or disease. The subject may have mild,intermediate or severe disease. The subject may be treatment naive,responding to any form of treatment, or refractory. The subject may bean individual in need of treatment or in need of diagnosis based onparticular symptoms or family history.

As used herein, the term “subject at risk” of getting a disease refersto estimating that a subject will have a disease or disorder in thefuture based on the subject's current symptoms, family history,lifestyle choices, and the like.

As used herein, the term “disease” refers to any condition, dysfunctionor disorder that damages or interferes with the normal function of acell, tissue, or organ.

The terms “measuring” and “determining” are used interchangeablythroughout, and refer to methods which include obtaining a subjectsample and/or detecting the level of a biomarker(s) in a sample. In oneembodiment, the terms refer to obtaining a subject sample and detectingthe level of one or more biomarkers in the sample. In anotherembodiment, the terms “measuring” and “determining” mean detecting thelevel of one or more biomarkers in a subject sample. Measuring can beaccomplished by methods known in the art and those further describedherein. The term “measuring” is also used interchangeably throughoutwith the term “detecting.”

Various methodologies of the presently disclosed subject matter includea step that involves comparing a value, level, feature, characteristic,property, and the like, to a “suitable control,” referred tointerchangeably herein as an “appropriate control” or a “controlsample.” A “suitable control,” “appropriate control” or a “controlsample” is any control or standard familiar to one of ordinary skill inthe art useful for comparison purposes. In one embodiment, a “suitablecontrol” or “appropriate control” is a value, level, feature,characteristic, property, and the like, determined in a cell, organ, orsubject, e.g., a control or normal cell, organ, or subject, exhibiting,for example, normal traits. For example, the biomarkers of the presentlydisclosed subject matter may be assayed for levels in a sample from anunaffected individual (UI) or a normal control individual (NC) (bothterms are used interchangeably herein). In another embodiment, a“suitable control” or “appropriate control” is a value, level, feature,characteristic, property, and the like, determined prior to performing atherapy (e.g., a cancer treatment) on a subject. In yet anotherembodiment, a transcription rate, mRNA level, translation rate, proteinlevel, biological activity, cellular characteristic or property,genotype, phenotype, and the like, can be determined prior to, during,or after administering a therapy into a cell, organ, or subject. In afurther embodiment, a “suitable control” or “appropriate control” is apredefined value, level, feature, characteristic, property, and thelike. A “suitable control” can be a profile or pattern of levels of oneor more biomarkers of the presently disclosed subject matter thatcorrelates to the presence of an aggressive and/or poorly differentiatedmetastatic cancer, to which a subject sample can be compared. Thesubject sample can also be compared to a negative control, i.e., aprofile that correlates to not having an aggressive and/or poorlydifferentiated metastatic cancer.

V. General Definitions

Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation. Unlessotherwise defined, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this presently described subject matter belongs.

Following long-standing patent law convention, the terms “a,” “an,” and“the” refer to “one or more” when used in this application, includingthe claims. Thus, for example, reference to “a subject” includes aplurality of subjects, unless the context clearly is to the contrary(e.g., a plurality of subjects), and so forth.

Throughout this specification and the claims, the terms “comprise,”“comprises,” and “comprising” are used in a non-exclusive sense, exceptwhere the context requires otherwise. Likewise, the term “include” andits grammatical variants are intended to be non-limiting, such thatrecitation of items in a list is not to the exclusion of other likeitems that can be substituted or added to the listed items.

For the purposes of this specification and appended claims, unlessotherwise indicated, all numbers expressing amounts, sizes, dimensions,proportions, shapes, formulations, parameters, percentages, parameters,quantities, characteristics, and other numerical values used in thespecification and claims, are to be understood as being modified in allinstances by the term “about” even though the term “about” may notexpressly appear with the value, amount or range. Accordingly, unlessindicated to the contrary, the numerical parameters set forth in thefollowing specification and attached claims are not and need not beexact, but may be approximate and/or larger or smaller as desired,reflecting tolerances, conversion factors, rounding off, measurementerror and the like, and other factors known to those of skill in the artdepending on the desired properties sought to be obtained by thepresently disclosed subject matter. For example, the term “about,” whenreferring to a value can be meant to encompass variations of, in someembodiments, ±100% in some embodiments ±50%, in some embodiments ±20%,in some embodiments ±10%, in some embodiments ±5%, in some embodiments±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from thespecified amount, as such variations are appropriate to perform thedisclosed methods or employ the disclosed compositions.

Further, the term “about” when used in connection with one or morenumbers or numerical ranges, should be understood to refer to all suchnumbers, including all numbers in a range and modifies that range byextending the boundaries above and below the numerical values set forth.The recitation of numerical ranges by endpoints includes all numbers,e.g., whole integers, including fractions thereof, subsumed within thatrange (for example, the recitation of 1 to 5 includes 1, 2, 3, 4, and 5,as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like)and any range within that range.

EXAMPLES

The following Examples have been included to provide guidance to one ofordinary skill in the art for practicing representative embodiments ofthe presently disclosed subject matter. In light of the presentdisclosure and the general level of skill in the art, those of skill canappreciate that the following Examples are intended to be exemplary onlyand that numerous changes, modifications, and alterations can beemployed without departing from the scope of the presently disclosedsubject matter. The synthetic descriptions and specific examples thatfollow are only intended for the purposes of illustration, and are notto be construed as limiting in any manner to make compounds of thedisclosure by other methods.

Example 1 Methods

Ethics statement: All animal experiments were conducted in accordancewith a protocol approved by the Johns Hopkins University Animal Care andUse Committee (protocol #MO11M270). Mice were housed in a sterileenvironment where they had free access to food and water as outlined inthe institutional guidelines.

Cell culture and proliferation assay: Cells (MDA-MB-231 and Hs578T) werecultured as recommended (ATCC). For proliferation assays, cells wereseeded (7,500/well) and counted using an automated cell counter(Nexcelom). Each experiment was done in triplicate and performed atleast twice.

RNA interference: The short-hairpin RNA interference vector for HMGA1targets 5′-CAACTCCAGGAAGGAAACCAA-3′ (SEQ ID NO:1) and has been describedelsewhere (Liau et al. (2006) Cancer Res. 66:11613-11622). Virus wasprepared as previously described. The empty vector was used as anegative control as described (Belton et al. (2012) PloS One 7:e30034).Polyclonal, transduced cells were selected and maintained in puromycin(1 ug/ml). To ensure that the effects of silencing HMGA were not aresult of a single clone, independent, polyclonal transductions weredone at least twice for each experiment. All functional experiments wereperformed in duplicate or triplicate and replicated after a repeattransduction experiment and polyclonal selection of shRNA or controlcells. Repression of HMGA1 was confirmed in each case at the level ofgene expression (qRT-PCR) and Western analysis.

Migration and invasion assays: Invasion assays were performed aspreviously described (Hillion et al. (2009) Mol. Cancer Res.7:1803-1812) with the following modifications. Briefly, 15,000 cellswere resuspended in serum-free media (500 ul) and placed in the upperchamber of a 24-well BD BioCoat™ Matrigel™ Invasion Chamber coated withMatrigel. Invasion was calculated as the percentage of total cells thatinvaded into the bottom chamber containing complete media with serum.Migration was performed similarly, except that Matrigel was omitted.

Orthotopic tumorigenicity and metastatic foci experiments: Cells(suspended in 75 ul phosphate-buffered saline (PBS)) were implanted intomurine (NOD-scid IL2Rgamme^(null)) mammary fat pads with an equal volumeof Matrigel. Tumor volumes (calculated by 4/3π×length/2×width/2×depth/2)were monitored daily until they reached 1-1.5 cm³, after which mice wereeuthanized. The presence of tumor foci within the lung was analyzedhistopathologically. For tail vein injection experiments, cells (10⁶)were resuspended in PBS (150 ul). Mice were euthanized after 3 weeks,and lungs were examined histopathologically.

Mammosphere assay: Mammosphere assays were performed as previouslydescribed (Shaw et al. (2012) J. Mammary Gland Biol. Neoplasia 17:111-117) and spheres (>50 um) were counted.

Western analysis: Western blots were performed as previously described(Belton et al. (2012) PloS One 7:e30034; Wood et al. (2000) Mol. Cell.Biol. 20:5490-5502), using commercial antibodies to HMGA1 (Abcam) andβ-Actin (Cell Signaling), both at a 1:1000 dilution.

Gene expression analysis with quantitative, reverse transcription PCR:Total RNA was isolated using the Direct-zol RNA MiniPrep kit (Zymo) andanalyzed by qRT-PCR as previously described. The expression level ofeach gene was normalized to the human RPLP0 (Applied Biosystems) orβ-actin gene. Primers for ECadherin, Snail, and Vimentin were previouslydescribed (Belton et al. (2012) PloS One 7:e30034; Mani et al. (2008)Cell 133:704-715).

HMGA1 knock-down and gene expression profile analysis: HMGA1 wasknocked-down in MDA-MB-231 cells using siRNA as previously described(Belton et al. (2012) PloS One 7:e30034; Tesfaye et al. (2007) CancerRes. 67:3998-4004; Hillion et al. (2009) Mol. Cancer Res. 7:1803-1812).RNA was isolated and hybridized to the Affymetrix exon array (GeneChipHuman Exon 1.0 ST Array) as previously described. Expression data waspreprocessed using the robust microarray (RMA) algorithm (Irizarry etal. (2003) Nucleic Acids Res. 31: el5), as implemented in the oligosoftware package (Carvalho and Irizarry (2010) Bioinformatics 26:2363-2367) available from Bioconductor (Gentleman et al. (2004) GenomeBiol. 5: R80) and annotated to the most recent human genome using thegetNetAffx function in the oligo package. Microarray data were uploadedto Gene Expression Omnibus (GSE45483). Expression profiles from HMGA1knock-down cells were compared to control cells treated with the vectorRNA using Bayes modified t-tests and the limma package from Bioconductor(Smyth (2005) “Bioinformatics and Computational Biology Solutions usingR and Bioconductor”, Springer-Verlag: 397-420). The analysis was focusedon the 100 most differentially expressed transcripts.

To compare this signature to genes expressed in a panel of embryonicstem cells, induced pluripotent stem cells, embryoid bodies, andfibroblasts, expression profiles were downloaded for a 43 sample study(GSE25970) (Bock et al. (2011) Cell 44: 439-452) from the GeneExpression Omnibus (Barrett et al. (2005) Nucleic Acids Res. 33:D562-D566). Unsupervised cluster analysis was performed on all probesannotated to the 63 genes in the HMGA1 panel using agglomorativeclustering with complete linkage. Euclidean distance and t-tests wereused to compare probe-specific expression in stem cells and fibroblasts.

Pathway analysis of differentially expressed genes was performed usingIngenuity Pathway Analysis (IPA, Ingenuity Systems) as described(Schuldenfrei et al. (2011) BMC Genomics 12:549). IPA scores weregenerated for each network and indicate the likelihood that the focusgenes present in the network could occur by chance alone. A score of ≧3is considered significant because it represents a 1/1,000 chance thatthe network contains specific focus genes by random chance alone(Schuldenfrei et al. (2011) BMC Genomics 12:549).

Results

Silencing HMGA1 halts cell proliferation and reprograms invasive,mesenchymal-like cells: To define the role of HMGA1 in oncogenicproperties and tumor progression, HMGA1 expression was silenced usinglentiviral-mediated delivery of short hairpin RNA (shRNA) ((Shah et al.(2012) PLoS One 7: e48533) in cell lines derived from aggressive,triple-negative breast cancers (MDAMB-231, Hs578T; FIG. 1A). Controlcells were transduced with a control lentiviral vector (Belton et al.(2012) PloS One 7:e30034; Shah et al. (2012) PLoS One 7: e48533). It wasdiscovered that cell proliferation was rapidly halted in both cell lines(FIG. 1B) within the first 4 days. Surprisingly, there was a dramaticchange in cell morphology whereby the spindle-shaped, fibroblast-likecells became cuboidal and epithelial-like in appearance (FIG. 1C).Because these morphologic changes are consistent with amesenchymal-epithelial transition, the expression of genes involved in amesenchymal-epithelial transition was investigated (Belton et al. (2012)PloS One 7:e30034; Mani et al. (2008) Cell 133:704-715). In MDA-MB-231cells, it was found that silencing HMGA1 led to a significant repressionin the mesenchymal genes, Snail and Vimentin, while there was anincrease in the gene expressing the epithelial marker, E-Cadherin (FIG.1D). Similarly, in Hs578T cells, E-Cadherin was induced when HMGA1 wassilenced. Tumor progression properties, including invasion andmigration, were also assessed. In both cell lines, there was a markedreduction in migration and invasion in cells with silencing of HMGA1(FIG. 1E). Together, these findings indicate that silencing HMGA1results in a profound decrease in proliferation, migration, andinvasion, as well as morphologic and gene expression changes consistentwith a mesenchymal-epithelial transition.

Silencing HMGA1 interferes with orthotopic tumorigenicity and metastaticprogression: Next, the role of HMGA1 on tumorigenesis was assessed usingin vivo models of triple-negative breast cancer. First, tumor growth wasassessed following mammary fat pad implantation. It was found thatsilencing HMGA1 in the aggressive MDA-MB-231 cells leads to a dramaticdecrease in tumor growth following mammary fat pad implantation (FIG.2A). Specifically, cells (10⁵) transduced with control virus reached avolume of 0.53 cm³±0.34 at 8 weeks following mammary fat padimplantation. In contrast, the tumors from cells transduced with HMGA1shRNA (shHMGA1) were significantly smaller at 8 weeks followingimplantation (0.037 CM³±0.058; p=0.016). Because there was a dramaticeffect on primary tumorigenesis, it was also determined if silencingHMGA1 interferes with metastatic progression. Therefore, the lungs wereevaluated histopathologically for tumor foci after necropsy. Strikingly,almost no metastatic lesions to the lungs in the mice implanted with theshHMGA1 cells were discovered as compared to the mice implanted withcontrol cells in which there were extensive, coalescing sheets ofmetastatic tumor cells throughout the lungs following mammaryimplantation with 10⁵ cells (FIG. 2B). Metastatic progression was alsoassessed following mammary fat pad implantation with a greater number ofcells (10⁷) from a repeat transduction experiment, and sacrificed themice after 5 weeks. With the higher number of cells, tumors formed fromall injections (3/3 in controls and 3/3 in shHMGA1 cells (FIG. 2C).Although tumors were slightly smaller from the shHMGA1 cells, thedifference was not significant (0.64±0.27 in controls versus 0.17±0.072in shHMGA1 cells, p=0.08). Despite the similar tumor volumes, a dramaticdecrease (>100-fold) was observed in metastatic foci in the shHMGA1cells as compared to controls (0.67±1.15 versus >100 in all controls;p=0.00004; FIGS. 2C-2D). Lung foci was also assessed following tail veininjection of control or shHMGA1 cells (10⁶) after 3 weeks. Only one lungfocus was observed after injection of the shHMGA1 cells, while therewere numerous foci in the control cells (0.25±0.5 versus 99.3±15.0;p=0.007; FIG. 5).

Silencing HMGA1 blocks mammosphere formation and depletestumor-initiator cells: Because silencing HMGA1 has profound effects ononcogenic properties in vitro, primary tumorigenesis and metastaticprogression in vivo, and expression of genes involved inepithelialmesenchymal transition, it was sought to determine its role incancer stem cell characteristics. To this end, the epithelial stem cellproperty of mammosphere formation (Shaw et al. (2012) J. Mammary GlandBiol. Neoplasia 17: 111-117) was explored in the control andshHMGA1-treated cells (FIGS. 3A-3B). It was found that growth ofprimary, secondary, and tertiary mammospheres was significantly impairedin the MDA-MB-231 cells with silencing of HMGA1. Similarly, it wasobserved that there was a significant decrease in primary mammosphereformation in the Hs578T cells treated with shHMGAl. (Secondary ortertiary mammospheres do not form in control Hs578T cells, precludinganalysis of these phenotypes). Next, orthotopic implantations wereperformed and tumorigenicity was assessed with limiting dilutions. Aspresented above, tumors formed in both control and shHMGA1 cells when10⁷ or 10⁵ cells were implanted. In contrast, no tumors formed in theMDA-MB-231 cells with silencing of HMGA1 when 10⁴ cells were injected(0/3), while tumors formed in all control injections (3/3; FIG. 3C).These results indicate that silencing HMGA1 in MDA-MB-231 cells depletesthe tumor-initiator or cancer stemlike cells and further underscores therole of HMGA1 as a key regulator of stem cell properties in aggressive,triple-negative breast cancer cells.

HMGA1 induces a stem cell signature in triple-negative breast cancercells: To globally define the transcriptional networks regulated byHMGA1, gene expression profile analysis was performed in MDAMB-231 cellswith or without HMGA1 knock-down. To this end, siRNA was used (Tesfayeet al. (2007) Cancer Res. 67:3998-4004; Hillion et al. (2009) Mol.Cancer Res. 7:1803-1812) and a rapid and significant reduction in HMGA1expression was observed (FIG. 6A). HMGA1 mRNA falls dramatically by 48hours, with persistent decreases at 72 hours (FIG. 6A). There was also amarked decrease in HMGA1 protein at 48 and 72 hours (FIG. 6B).Therefore, global gene expression profile analysis was performed at 48hours using an Affymetrix exon array (GeneChip Human Exon 1.0 ST Array)with RNA from three independent replicates of each experimentalcondition. To define an HMGA1 signature in breast cancer, 100transcripts that were most differentially expressed were identified.These 100 transcripts correspond to 63 unique genes. Because HMGA1 isenriched in embryonic stem cells and functional studies showed that itis required for cancer stem cell properties, the HMGA1 signature of 63genes was compared to gene expression profiles from diverse pluripotentstem cells and differentiated cells, including embryonic stem cells(ESCs), induced pluripotent stem cells, embryoid bodies, and fibroblasts(Bock et al. (2011) Cell 44: 439-452). As shown, un-supervised clusteranalysis of these genes separates the samples by cell type with a cleardistinction between pluripotent stem cells and differentiated cells.Moreover, the HMGA1 signature is highly enriched inpluripotent/embryonic stem cells (p<0.001; FIG. 4A). A subset of theHMGA1 signature genes was validated using quantitative RT-PCR, anddifferential expression similar to the microarray gene expressionresults was found in all cases (FIG. 6C). These findings suggest thatHMGA1 drives tumor progression by inducing stem cell transcriptionalnetworks.

To elucidate cellular pathways regulated by HMGA1 in breast cancer, theHMGA1 signature was analyzed with Ingenuity Pathway Analysis (IPA,Ingenuity Systems). From the top list of differentially regulated genes,two pathways had significant network scores (69 and 46, respectively;FIGS. 4B and 7). The highest scoring network was embryonic development,tissue development, and cellular development. The top molecular andcellular functions were cell death and survival and cellular movement,while the top physiologic system development and functions included: 1.nervous system development and function, 2. organ morphology, and 3.embryonic development. In this network, the most down-regulated moleculewas ARL2BP or ADP-ribosylation factor (ARF)-like 2 binding protein. Thisprotein is a member of a functionally distinct group of RAS-relatedGTPases, called the ARF family. ARL2BP protein binds to ARL2.GTP withhigh affinity and plays a role in the nuclear translocation, retentionand transcriptional activity of STAT3 (Muromoto et al. (2008) Int.Immunol. 20: 395-403). Notably, it was shown that HMGA1 induces STAT3expression in lymphoid tumorigenesis, and STAT3 inhibitors are cytotoxicto the HMGA1-driven tumor cells (Hillion et al. (2008) Cancer Res.68:10121-10127). TMCO1 or transmembrane and coiled-coil domains 1protein was the most up-regulated protein in this network. Although itsfunction is not known, it is associated with breast cancer cells[8882811 GEO Profiles-NCBI]. TGFβ1 is a major node and this protein isupregulated in diverse cancers and thought to promote invasion,migration, EMT and tumor progression (Massague (2008) Cell 134:215-230). EGFR and MAPK are other important nodes that are activated incancer and mediate proliferative signals (Schuldenfrei et al. (2011) BMCGenomics 12:549). Another central node was HIF-1 alpha, a key factorinvolved in angiogenesis during tumor progression and vasculardevelopment during embryogenesis (Semenza (2012) Oncogene in press. Inaddition, Myc was identified as a major node and prior studies foundthat not only does cMYC induce HMGA1 expression (Wood et al. (2000) Mol.Cell. Biol. 20:5490-5502), but HMGA1 also directly up-regulates cMYCexpression (Shah et al. (2012) PLoS One 7: e48533). Myc also has awell-defined role in breast (Thibodeaus et al. (2009) Breast Cancer Res.Treat. 116:281-294; Tront et al. (2010) Cancer Res. 70:9671-9681) andother diverse cancers (Dang (2012) Cell 2:304-307) as well as inembryonic stem cells (Dang (2012) Cell 2:304-307; Nie et al. (2012) Cell151:68-79. Thus, this pathway analysis further confirms the importantrole for HMGA1 in regulating embryonic stem cell networks during tumorprogression in breast cancer.

Discussion

The presently disclosed subject matter reports for the first time thatsilencing HMGA1 induces a rapid and dramatic reprogramming of highlyproliferative, invasive, mesenchymal-like breast cancer cells to moredifferentiated, slowly growing, epithelial-like cells. It was found thatknock-down of HMGA1 has profound effects on oncogenic propertiesassociated with both tumor initiation (orthotopic tumorigenesis) andtumor progression (migration, invasion, and metastatic progression). Infact, the in vivo effects on metastatic progression were even morepronounced than the effects on primary tumorigenesis, thus highlightingthe role of HMGA1 in tumor progression. The changes induced by silencingHMGA1 are among the most striking alterations reported to date withknockdown of HMGA1 or most other oncogenes for that matter, both indegree and rate of onset. The profound effects could be related to thepresently disclosed efficient, viral-mediated delivery of shRNA torepress HMGA1. In addition, triple-negative breast cancer cells may behighly dependent upon HMGA1 and related pathways for their oncogenicproperties. Indeed, a study from the Broad Institute at MIT identifiedHMGA1 as a key transcription factor enriched in triple-negative breastcancer (Ben-Porath et al. (2008) Nat. Genet. 40:499-507). Moreover,expression of HMGA1 and 8 additional genes predicted poor outcomes inbreast cancer, as well as brain and bladder cancer. Prior studies usingantisense or dominant-negative approaches in triple-negative breastcancer cells (MDA-MB-231 or Hs578T) also showed thatanchorage-independent cell growth or colony formation are inhibited byHMGA1 repression (Reeves et al. (2001) Mol. Cell. Biol. 21:575-594;Dolde et al. (2002) Breast Cancer Research and Treatment 71:181-191).There is also preliminary evidence demonstrating that HMGA1 expressioncorrelates with more advanced nuclear grade in primary tumors (Asch &Resar, unpublished data).

Emerging evidence further indicates that HMGA1 is important inmaintaining a de-differentiated, pluripotent stem-like state ((Shah etal. (2012) PLoS One 7: e48533). A recent landmark paper demonstratedthat HMGA1 is required for cellular reprogramming of somatic cells toinduced pluripotent stem cells (iPSCs) by the Yamanaka factors (Flohr etal. (2003) Histol. Histopathol. 18: 999-1004). Blocking HMGA1 expressionor function prevents the derivation of iPSCs. In normal embryonic stemcells in culture and during the reprogramming process to iPSCs, HMGA1activates expression of stem cell transcriptional networks. Recentstudies also found that tumor progression and an epithelial-mesenchymaltransition (EMT) involves transcriptional networks important in stemcells (Ben-Porath et al. (2008) Nat. Genet. 40:499-507; Schuldenfrei etal. (2011) BMC Genomics 12:549; Belton et al. (2012) PloS One 7:e30034;Shah et al. (2012) PLoS One 7: e48533; Mani et al. (2008) Cell133:704-715). The first evidence linking HMGA1 to EMT came from animportant study in 2001 in MCF-7 breast cancer cells, which demonstratedthat forced expression of HMGA1 results in metastatic progression andhistologic changes consistent with EMT in the epithelial MCF-7 breastcancer cell line (Dolde et al. (2002) Breast Cancer Research andTreatment 71:181-191). This group also found that HMGA1 induces changesin classes of genes involved in tumor progression. More recently,studies in colon cancer showed that HMGA1 is required for tumorprogression and stem cell properties (Belton et al. (2012) PloS One7:e30034). The presently disclosed subject matter shows that HMGA1 isrequired for mammosphere formation, including secondary and tertiarymammospheres in MDA-MB-231 cells. It has also been found that silencingHMGA1 depletes tumor initiator/cancer stem cells, indicating thattargeting HMGA1 in breast cancer therapy could have an important impacton the cancer stem cell population, which is believed to be the basisfor refractory disease in diverse tumors. These functional studies arecorroborated by the HMGA1 signature and pathway analysis demonstratingthat HMGA1 orchestrates transcriptional networks important in stem cellsand metastatic progression.

There is a dire need to understand the molecular underpinnings ofmetastatic progression because this is the major cause of death inpatients with cancer. Although cancer is a highly complex andheterogeneous disease, with significant heterogeneity even within asingle tumor, increasing evidence indicates that common, centralpathways exist that could serve as “Achilles heels” or rationaltherapeutic targets in diverse tumors. The presently disclosed subjectmatter underscores the fundamental role for HMGA1 in tumor progressionin preclinical models for aggressive, triple-negative breast cancers andprovides compelling evidence that HMGA1 is a master regulator in theevolution of primary tumors to metastatic disease.

Example 2

The HMGA1 gene encodes the HMGA1a and HMGA1b chromatin remodelingproteins, which regulate gene expression by altering chromatinstructure. HMGA1 plays a central role in the development and progressionof PDAC by acting as a master regulator of transcriptional networks thatmaintain tumor cells in a refractory, stem-like state (FIG. 8 and FIG.9). It has been discovered that HMGA1 is overexpressed in >90% of PDACswith absent levels in precursor lesions and normal tissue. Moreover,previous work demonstrates that high levels of HMGA1 protein correlatepositively with poor differentiation status and decreased survival. Itwas also found that HMGA1 is required for reprogramming somatic cells toinduced pluripotent stem cells by the Yamanaka factors. In addition, itwas discovered that HMGA1 induces stem cell transcriptional networks incancer cells and normal stem cells. Taken together, these studiessuggest that HMGA1 drives poor differentiation and metastaticprogression in PDAC by inducing stem cell genes. Previous studiesindicate that knock-down of HMGA1 blocks oncogenic phenotypes in vitroand metastatic progression in orthotopic murine models. Knock-down ofHMGA1 also blocks cancer stem cell properties and depletes cancer stemcells/tumor initiator cells.

Results

Silencing HMGA1 with short hairpin RNA (shRNA) reprograms PDAC cells:HMGA1 and HMGA2 expression levels in different pancreatic ductaladenocarcinoma cell (PDAC) lines are shown in FIG. 11. In low-passagePDAC cells derived from patient tumors (lines 10.7, 10.05) or MiaPaCa2commercial PDAC cell lines, it was discovered that silencing HMGA1 usingviral-mediated delivery of shRNA drastically impairs cell growth (FIG.10 and FIG. 12). Surprisingly, there is also a dramatic change inmorphology, with fibroblast-like cells becoming cuboidal orepithelial-like, consistent with a mesenchymal-epithelial transition, orMET (FIG. 11 and FIG. 14;) Preliminary studies also show that colonyformation, migration, and invasion are disrupted in the HMGA1 knock-downcell. (FIG. 13 and FIG. 15B). In preliminary experiments, xenografttumor formation was inhibited in the HMGA1 shRNA cells. Strikingly, itwas also found that silencing HMGA1 blocks stem cell properties,including 3-dimensional (3-D) sphere formation in PDAC cells (FIG. 15C).HMGA1 knock-down also prevents tumor formation following injection ofreduced numbers of cells or “limiting dilutions” in patient-derived PDACcells; studies are underway to more precisely calculate the frequency oftumor-initiator cells in the control and wild type PDAC cells. Thislatter finding indicates that the tumor initiator/cancer stem cells(CSCs) are depleted in the HMGA1 knock-down cells. Similar findings wereobserved in other cancer cells treated with shRNA targeting HMGA1,including colorectal cancer (CRC) and leukemia cells (FIG. 16A, FIG. 16Band FIG. 16C). Tumorigenicity and additional oncogenic assays areunderway in PDAC cells. Gene expression analysis is also planned toidentify the molecular pathways that are disrupted after silencingHMGA1.

Systemic nanoparticle (NP) plasmid delivery of shRNA to HMGA1: Theexperiments shown herein demonstrate that silencing HMGA1 in PDAC cellsis an effective anti-tumor therapy. Innovative NP platforms are beingtested to systemically deliver shRNA plasmid DNA in vivo in preclinicalanimal models. It was found that there was a decrease in average tumorsize in tumors treated with NP delivery of plasmid DNA vectors encodingshRNA to target HMGA1 as compared to control NPs (FIG. 17). Onhistologic examination, necrosis appeared to be increased in the tumorstreated with the HMGA1-targeting vector (FIG. 18A and FIG. 18B).Fluorescent-activated cell sorting (FACS) analysis following injectionof labeled DNA, however, showed that the plasmid was delivered to only˜5-30% of cells.

Discussion

Together, these results show that silencing HMGA1 reprograms aggressivestem-like cancer cells into non stem-like cells with slow growth andaltered properties. Results demonstrated that silencing HMGA1 hasdramatic effects on tumor cell appearance, proliferation and invasiveproperties. It was also found that silencing HMGA1 prevents 3D sphereformation and depletes cancer stem cells. Silencing HMGA1 also haltsproliferation and blocks hallmarks of aggressive PDA includingself-renewal, migration, invasion, and anchorage independent cellgrowth. It is expected that nanoparticle delivery of specificallytargeted RNA inhibitors will function as viable therapeutic approachesto aggressive neoplastic cancer. In addition, preliminary studiessuggest that NP delivery of plasmid DNA expressing shRNA to target HMGA1can be delivered to xenograft tumor cells and may impair growth.

REFERENCES

All publications, patent applications, patents, and other referencesmentioned in the specification are indicative of the level of thoseskilled in the art to which the presently disclosed subject matterpertains. All publications, patent applications, patents, and otherreferences are herein incorporated by reference to the same extent as ifeach individual publication, patent application, patent, and otherreference was specifically and individually indicated to be incorporatedby reference. It will be understood that, although a number of patentapplications, patents, and other references are referred to herein, suchreference does not constitute an admission that any of these documentsforms part of the common general knowledge in the art.

Although the foregoing subject matter has been described in some detailby way of illustration and example for purposes of clarity ofunderstanding, it will be understood by those skilled in the art thatcertain changes and modifications can be practiced within the scope ofthe appended claims.

That which is claimed:
 1. A method of inhibiting at least one cancerstem cell, the method comprising contacting the at least one cancer stemcell with an effective amount of at least one HMGA 1 inhibitor.
 2. Themethod of claim 1, wherein inhibiting the at least one cancer stem cellis selected from the group consisting of: i) inhibiting proliferation ofthe at least one cancer stem cell; ii) inhibiting self-renewal of the atleast one cancer stem cell; iii) inhibiting anchorage-independent growthof the at least one cancer stem cell; iv) inhibiting migration of the atleast one cancer stem cell; v) inhibiting invasion of the at least onecancer stem cell; vi) reprogramming the at least one cancer stem cellfrom a stem-like state that is refractory to apoptosis to a nonstem-like state that is susceptible to apoptosis; and vii) combinationsthereof.
 3. The method of claim 1, wherein the at least one cancer stemcell is in an aggressive and/or poorly differentiated metastatic tumor,and inhibiting the at least one cancer stem cell inhibits at least oneof: i) growth of the aggressive and/or poorly differentiated metastatictumor; ii) proliferation of the aggressive and/or poorly differentiatedmetastatic tumor; iii) migration of the aggressive and/or poorlydifferentiated metastatic tumor; iv) invasion of the aggressive and/orpoorly differentiated metastatic tumor; v) initiation of new aggressiveand/or poorly differentiated metastatic tumors; and vi) combinationsthereof.
 4. The method of claim 1, wherein the at least one cancer stemcell expresses greater levels of HMGA1 as compared to non-stem cancercells in the aggressive and/or poorly differentiated metastatic tumor.5. The method of claim 1, wherein the at least one cancer stem cell isselected from the group consisting of a triple-negative breast cancercell, a pancreatic ductal adenocarcinoma cell, a colorectal cancer cell,and a leukemia cell.
 6. The method of claim 1, wherein the at least oneHMGA1 inhibitor reduces the expression level and/or activity of HMGA1.7. The method of claim 1, wherein the at least one HMG1 inhibitor is anRNA interfering agent.
 8. The method of claim 7, wherein the at leastone HMGA1 inhibitor is an shRNA.
 9. The method of claim 8, wherein theshRNA targets the nucleotide sequence of SEQ ID NO:1.
 10. The method ofclaim 1, further comprising contacting the at least one cancer stem cellwith a chemotherapeutic agent.
 11. The method of claim 10, wherein thechemotherapeutic agent is gemcitabine.
 12. The method of claim 1,wherein the at least one cancer stem cell is contacted in a subject. 13.The method of claim 12, wherein the subject is a human subject.
 14. Amethod of treating an aggressive and/or poorly differentiated metastaticcancer in a subject in need thereof comprising administering atherapeutically effective amount of at least one HMGA1 inhibitor to thesubject.
 15. The method of claim 14, wherein the aggressive and/orpoorly differentiated metastatic cancer is selected from the groupconsisting of triple-negative breast cancer, pancreatic ductaladenocarcinoma cell, colorectal cancer cell, and leukemia.
 16. Themethod of claim 14, wherein the aggressive and/or poorly differentiatedmetastatic cancer comprises at least one cancer stem cell thatoverexpresses HMGA1 protein.
 17. The method of claim 14, furthercomprising selecting the subject for treatment of the aggressive and/orpoorly differentiated metastatic cancer with the at least one HMGA 1inhibitor.
 18. The method of claim 17, wherein selecting the subject fortreatment of the aggressive and/or poorly differentiated metastaticcancer comprises: (i) obtaining a biological sample comprising cellsfrom the aggressive and/or poorly differentiated metastatic cancer; (ii)assaying the level of HMGA1 expression in the cells from the aggressiveand/or poorly differentiated metastatic cancer; (iii) comparing thelevel of HMGA1 expression in the cells to the level of HMGA1 expressionin a normal control cell; and (iv) selecting the subject for treatmentof the aggressive and/or poorly differentiated metastatic cancer withthe at least one HMGA1 inhibitor if the level of HMGA1 expression in thecells is greater than the level of HMGA1 expression in the normalcontrol cell.
 19. The method of claim 18, wherein the biological sampleis selected from the group consisting of a breast tissue sample, apancreatic tissue sample, a colon tissue sample, and a bone marrowtissue sample.
 20. The method of claim 18, wherein at least some of thecells from the aggressive and/or poorly differentiated metastatic cancercomprise cancer stem cells.
 21. The method of claim 14, wherein the atleast one HMGA1 inhibitor reduces the expression level and/or activityof HMGA1.
 22. The method of claim 14, wherein the at least one HMGA1inhibitor is formulated for delivery in a nanoparticle.
 23. The methodof claim 14, wherein the at least one HMG1 inhibitor is an RNAinterfering agent.
 24. The method of claim 23, wherein the at least oneHMGA1 inhibitor is an shRNA.
 25. The method of claim 24, wherein theshRNA targets the nucleotide sequence of SEQ ID NO:1.
 26. The method ofclaim 24, wherein the shRNA is formulated for delivery in ananoparticle.
 27. The method of claim 14, wherein the subject is a humansubject.
 28. The method of claim 14, further comprising administering aneffective amount of a chemotherapeutic agent to the subject.
 29. Themethod of claim 28, wherein the chemotherapeutic agent is gemcitabine.30. A method of selecting a subject with an aggressive and/or poorlydifferentiated metastatic cancer for treatment with at least one HMGA1inhibitor, the method comprising: (a) obtaining a biological sample fromthe subject, wherein the biological sample comprises cells from theaggressive and/or poorly differentiated metastatic cancer; (b)determining the level of expression of HMGA1 in the biological sample;(c) comparing the level of expression of HMGA1 in the biological samplewith the level of expression of HMGA1 in a control sample; and (d)selecting the subject with aggressive and/or poorly differentiatedmetastatic cancer for treatment with at least one HMGA1 inhibitor whenthe level of HMGA1 expression in the biological sample is greater thanthe level of HMGA1 expression in the control sample.
 31. The method ofclaim 30, wherein the biological sample is selected from the groupconsisting of a breast tissue sample, a pancreatic tissue sample, acolon tissue sample, and a bone marrow tissue sample.
 32. The method ofclaim 30, wherein the aggressive and/or poorly differentiated metastaticcancer is selected from the group consisting of triple-negative breastcancer, pancreatic ductal adenocarcinoma cell, colorectal cancer cell,and leukemia.
 33. The method of claim 30, wherein the subject is a humansubject.
 34. The method of claim 30, further comprising treating thesubject with aggressive and/or poorly differentiated metastatic cancerby administering an effective amount of at least one HMGA1 inhibitor tothe subject.
 35. The method of claim 34, further comprisingadministering an effective amount of at least one chemotherapeutic agentto the subject.